|Year : 2019 | Volume
| Issue : 64 | Page : 366-370
Naphthazarins as cytotoxic agents isolated from Arnebia euchroma
Nidhi Sharma1, Ashu Gulati2, Dharmesh Kumar3, Yogendra Padwad3
1 Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Road Research Institute, Delhi-Mathura Road, CRRI, New Delhi; Food and Nutraceuticals Division, Natural Product Chemistry Lab, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
2 Food and Nutraceuticals Division, Natural Product Chemistry Lab, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
3 Food and Nutraceuticals Division, Pharmacology and Toxicology Lab, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
|Date of Submission||14-Mar-2019|
|Date of Decision||18-May-2019|
|Date of Web Publication||23-Aug-2019|
Food and Nutraceutical Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur - 176 061, Himachal Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Currently, many compounds of different medicinal plants are under investigation for curing various types of cancers. In extension with this ongoing research, Arnebia euchroma is taken for the study of cytotoxic compounds present in its roots. The sensitivity of human cervical cancer (SiHa), human epidermoid carcinoma (KB), and human colorectal cancer 116 (HCT116) cell lines toward C1 and C2 isolated from A. euchroma roots was evaluated. Materials and Methods: The root extract of A. euchroma was subjected to extraction, chromatographic separation to afford two naphthazarin esters C1 and C2 as well as two white-colored compounds. The structures of purified compounds were determined by electrospray ionization mass spectrometry and nuclear magnetic resonance analysis. Results: Both C1 and C2 showed potent cytotoxicity against SiHa, KB, HCT116 cells at different concentrations. Conclusion: C1 and C2 exhibited strong cytotoxicity with IC50= 0.02 μM and 0.09 μM against SiHa cells and with IC50= 0.11 μM HCT116 cells than the vinblastine standard, respectively.
Keywords: Arnebia euchroma, cell lines, cytotoxicity, dietary supplements, naphthazarin esters
|How to cite this article:|
Sharma N, Gulati A, Kumar D, Padwad Y. Naphthazarins as cytotoxic agents isolated from Arnebia euchroma. Phcog Mag 2019;15, Suppl S2:366-70
|How to cite this URL:|
Sharma N, Gulati A, Kumar D, Padwad Y. Naphthazarins as cytotoxic agents isolated from Arnebia euchroma. Phcog Mag [serial online] 2019 [cited 2019 Sep 16];15, Suppl S2:366-70. Available from: http://www.phcog.com/text.asp?2019/15/64/366/265014
- Two naphthazarin esters C1 (hydroxyisovalerylshikonin) and C2 (acetylshikonin) were isolated and purified from Arnebia euchroma roots. The cytotoxic action of C1and C2 was studied against human cervical cancer (SiHa), human epidermoid carcinoma (KB), and human colorectal cancer 116 (HCT 116) cell lines. The cytotoxicity of test compounds was evaluated using sulforhodamine B assay. In the results, C1 showed effective 93.4% ± 3.8% cell inhibition at the concentration of 0.25 μM and C2 showed 88.4% ± 2.5% cell inhibition at the concentration of 0.15 μM and 100.0% ± 0.2% cell inhibition at the concentration of 0.30 μM on HCT116 cell line. Further, it is found that the presence of an acetyl group may be decisive for the cytotoxicity, as C2 exhibited higher % cell inhibition on SiHa, KB, and HCT 116 cancer cell lines than C1. The C2 showed 100.0% ± 0.2% cell inhibition at a concentration of 0.30 μM on HCT116 cell line than C1 that showed 93.4% ± 3.8% at a concentration of 0.25 μM, respectively.
Abbreviations used: SiHa cells: Human cervical cancer cells; KB cells: Human epidermoid carcinoma cells; HCT116 cells: Human colorectal cancer cells; Ppm: Parts per million; μL: Microliter; μM: Micromolar; TLC: Thin-layer chromatography; ESI-MS: Electrospray ionization-mass spectrometry; TCM: Traditional Chinese medicine.
| Introduction|| |
Naphthazarin esters are well-known potent pharmaceutical compounds present in plants of genus Arnebia. Among various Arnebia species, Arnebia euchroma (Royle ex Benth.) is a perpetual member in the family Boraginaceae. It is widely distributed in the Alpine Belt and the Himalayas. It is a herbaceous perennial plant. A. euchroma has thick roots of diameter 2 cm, a cluster of basal leaves and several flowering stems up to 50 cm tall. It was listed in the 2005 Chinese Pharmacopoeia as the main resource of medicinal substances Zicao. Zicao has been used for the treatment of throat sores, burns, cuts, and skin diseases such as macular eruption, measles, and carbuncles in China back from the fifth century. Previous phytochemical studies on this plant reported the isolation of naphthoquinones monoterpenes, phenols, organic acids, and pyrrolizidine alkaloids.,,,,,,,, Shikonin and its derivatives are naphthazarin esters of hydroxynaphthoquinones that are present as ester derivatives in the outer surface of the roots in Arnebia species. Naphthazarin esters are used as natural colorants for food, cosmetics, and textiles. These naphthazarin are reported to have anti-inflammatory, antifungal, antioxidant, cytotoxic, and radical scavenging activities and enzyme inhibitory properties.,,,,,, Further, acyl derivatives of isohexenylnaphthazarin have been investigated for topoisomerase I inhibition and proved to be potential anticancer agents.,,, Furthermore, the increasing problem of cancer in the developed world, leading to death has motivated the extensive growth of cancer research in recent years. Mass screening programs of natural products and synthetic compounds by the National Cancer Institute of the USA have identified the Quinone moiety as a pharmacophore that normally affords the cytotoxic activity. These results provided a framework to study other shikonin derivatives as promising compounds for cytotoxicity assays. Thus, further research in the field is certain to continue for the emergence of a clinically useful anticancer agent. Moreover, accumulating research evidence suggests that many dietary factors may be used alone or in combination with traditional chemotherapeutic agents to prevent the occurrence of cancer, their metastatic spread, or even to treat cancer. As naphthazarin are well-known dietary supplements with accepted antioxidant behavior, based on earlier reported literature on antioxidant and cytotoxic nature of naphthazarin, the study presented the potent percentage cell inhibition by naphthazarin esters hydroxyisovalerylshikonin and acetylshikonin against Human cervical cancer (SiHa), human epidermoid carcinoma (KB), human colorectal cancer 116 (HCT116) cell lines.
| Materials and Methods|| |
The roots of A. euchroma were collected from Lahaul and Spiti Districts of Himachal Pradesh in Western Himalayas of India. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE III 600 MHz for 1 H and 13 C NMR, and chemical shifts were reported in parts per million δ downfield from internal standard Me4 Si (TMS) [Figures S1-4]. Multiplicities of signals are described as follows: S – singlet, br. S – broad singlet, d – doublet, t – triplet, and m – multiplet. Mass spectrums were on recorded Q-time-of-flight micromass liquid chromatography-mass spectrometry (MS) spectrometer [Figures S5 and 6].
Extraction and isolation of compounds
The roots of A. euchroma (100 g) was air-dried under shade temperature 27°C ± 2°C and relative humidity of 34% ± 3% for 5 days before extraction and analysis. The roots were powdered and exhaustively extracted with ethyl acetate (boiling point 60°C–80°C). The polar solvent extract was concentrated under vacuum to give a reddish-brown viscous residue (13.8 g). The extract was chromatographed on a column of Si gel and eluted with n-hexane in ethyl acetate (0%–100% gradient) and methanol to afford four fractions (C1–C4). Out of the four fractions, C3 and C4 are white colored fractions. The remaining two fractions C1and C2 were ultraviolet (UV) absorbing fractions with semi-viscous dark reddish colored and dry light red colored, respectively [Figure 1]. Initial separation and purification of different fractions were checked on normal phase Si gel thin layer chromatography (TLC) plate developed in solvent systems of hexane ethyl acetate in the ratio ranging from 0:100 to 100:0.
Cell lines and cell culture
SiHa and KB cell lines were obtained from the National Centre for Cell Science, Pune, India. HCT116 cell line, was obtained from the Indian Institute of Integrative Medicine, Jammu. All the cell lines were cultured in Dulbecco's Modified Eagle Medium (Invitrogen Biosciences, India), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Biosciences, India) and 1% antibiotic-antimycotic solution (Invitrogen Biosciences, India). The cell lines were maintained at 37°C in the CO2 incubator.,,
Sulforhodamine B assay
Cell lines were trypsinized and washed twice with phosphate buffered saline by centrifugation. SiHa and KB cells incubated at a density of 2 × 104 cells/well and HCT116 cells were incubated at a density of 1 × 104 cells/well in 96 well plates in 100 μL complete medium. Several dilutions (10, 25, 50, and 100 μg/mL) of the test compounds (in 100 μL of complete medium) were added. Vinblastine (1 μM) was used as a positive control, whereas cells alone supplemented with the complete medium used as a negative control. Plates were incubated at 37°C for 48 h in a CO2 incubator. After 48 h, 50 μL of 50% trichloroacetic acid (Sigma Aldrich, India) was added, and the plates were kept at 4°C for 1 h. The plates were flicked and washed five times with water and then air-dried. Subsequently, 100 μL of sulforhodamine B (SRB) solution (Sigma Aldrich, India) was added and incubated for 30 min at room temperature. After incubation, plates were washed five times with 1% acetic acid, air dried. 100 μL of 10 mM Tris base (Sigma Aldrich, India) was added. The absorbance was measured using a microplate reader (BioTeK Synergy H1 Hybrid Reader) at a wavelength of 540 nm. The IC50 values were calculated by “Quest Graph™ IC50 Calculator” AAT Bioquest, Inc.,
| Results and Discussion|| |
The similar fractions of purified A. euchroma root extract were collected and combined by repeated TLC experiments, to afford purified compounds C1–C2.1 H and 13 C NMR and electrospray ionization (ESI)-MS studies characterized compounds C1–C2 [Table 1] and Figures S1-6]. In the 1 H-NMR spectra of C1 and C2, the peaks belonging to side chains (C1´–C6´) were similar to each other. Compound 1 obtained as the red-colored powder was named as hydroxyisovalerylshikonin. UV spectrum of 1 showed λmax at 214, 274, 485, 518, and 559 nm. Its molecular formula was deduced as C21H24O7 by ESI-MS (m/z 388, 389 [C1+H], 411 [C1+Na]). There are 21 signals in the C13 spectrum in which peaks at δC1178.2, δC2136.4, δC3147.5, δC4178.3, δC5167.3, δC8166.9, δC6-7132.1 and 132.6, δC9-10111.6 implied that C1 was naphthazarin ester derivative of hydroxynaphthoquinones. The 1 H and 13 C NMR spectra of C1 showed typical phenolic OH groups at δH12.24 and δH12.42. Characteristic signals of aromatic H6-H7 and H3 protons of naphthazarin moiety are seen at δH7.13, δC132.1, δC132.6 and δH6.90, δC136.40, respectively. In addition to the singlets of methyl protons, H5' and H6´ at δH1.59 and 1.50, δC17.9, 25.7 and H4” and H5” at δH1.18, 1.11, δC28.7 the 1 H NMR spectrum displayed methylene protons H2' at δH2.06–2.24 (multiplet, bd, J = 3 Hz,) δC32.8 and H2” at δH2.42–2.58 multiplet, δC46.4, H3' at δH5.08 (triplet, J = 7.0 Hz, δC123.0) and H1at δH5.90 (1H, multiplet) δC70.7. The structure of hydroxyisovaleryl chain (C3-O3-H9) and its connectivity to naphthoquinone ring in the molecular structure was further confirmed by the observed signals at δH3.21 (s, OH) and δH2.42–2.58 (2H, multiplet) δC46.4. Compound 2 obtained as the red-colored powder was named as acetyl shikonin. UV spectrum of 2 showed λmax at 213, 273.50, 483, 495, and 562.00 nm. Its molecular formula was deduced as C18H18O6 by ESI-MS (m/z 330, 331 [C2+H], 353 [C2+Na]). There are 15 signals in the C13 spectrum in which peaks at δC1175.7, δC2130.8, δC3147.5, δC4177.3, δC5167.8, δC6-7132.4, and 132.7 shows that C2 was ester derivative of hydroxynaphthoquinones. The 1 H NMR spectra of 2 showed typical phenolic OH groups at δH12.22 and δH12.38. Characteristic singlets of aromatic H6-H7 and H3 protons of naphthazarin moiety are seen at δH7.09, δC132.4, 132.7 and δH6.88, δC130.84, respectively. In addition to the singlets of methyl protons H5 and H6 at δH1.44-1.55, δC16.58, 24.48 and the 1 H NMR spectrum displayed methylene protons H2' at δH2.34–2.46, multiplet, δC32.42 and H2” at δH1.99, 19.42, H3' at δH5.03 triplet, δC117.7 and H1' at δH5.86 (1H, quartet) δC69.40 [Table 1].
The cytotoxicity of C1and C2 against SiHa, KB, and HCT116 cell lines was evaluated using the SRB assay. These two compounds were tested for cytotoxicity at different concentrations (10, 25, 50, and 100 μg/mL) against the entire cell lines with vinblastine as a positive control. Results are shown in [Table 2]. C1 was effective with IC50= 0.02 μM against SiHa cell lines. It displays inhibition on KB cells with IC50= 0.30 μM and against HCT116 cells with IC50= 0.23 μM. C2 displays significant inhibition on SiHa cells with IC50= 0.096 μM and with IC50= 0.36 μM against KB-cell lines. It showed notable inhibition against HCT116 cells with IC50= 0.11 μM, respectively. Both the compounds come up as potent scavengers against all the three-cell lines as compare to positive control vinblastine [Figure 2], [Figure 3], [Figure 4] and [Table S1].
|Table 2: IC50 values (uM) of compounds C1-C2 against human cervical cancer, human epidermoid carcinoma, and human colorectal cancer 116 human cancer cell lines|
Click here to view
As shown in [Table 2], C1 showed prominent inhibitory concentration with IC50= 0.02 μM against SiHa cell lines and C2 prominent inhibition against SiHa cells with IC50= 0.09 μM and HCT116 cells with IC50= 0.11 μM. Similar cytotoxic results from callus and cell suspension cultures of A. euchroma were reported by Damianakos et al. Authors from this study tested the cytotoxic activity of acetylshikonin and hydroxyisovalerylalkannin against HCT116 cells along with other cell lines in 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) viability assay. While acetylshikonin shows inhibition with IC50 of 9.02 μM, hydroxyisovalerylkannin depicted cytotoxic effect at IC50= 12.50 μM against HCT116 cells. Moreover, these compounds were reported to inhibit human cancer cell lines without or little effect on normal cells.,, The nontoxicity of compounds further compliments their use as selective cytotoxic drugs against specific cancer cell lines. There are some mechanisms, which are proposed for the cytotoxic action of naphthoquinones such as oxidative stress, which arises from the capacity of these compounds to enter into redox cycle to generate-free radicals. The generation of free radicals may also cause cellular damage. The inhibition of enzymes by quinones vital for replication may be the other cause. Ahn and coworkers identified structure-activity relationships of these derivatives to find their order of capturing cellular nucleophiles as 6-substituted derivatives >2-substituted derivatives >naphthazarin. The most reasonable mechanism behind the cytotoxicities of C1 and C2 proposed based on the above results is bioreductive alkylation. C1 and C2 quinones are substituted with a side-chain bearing good leaving group (X) hydroxyisovaleryl in C1 and acetyl in C2 at the 2-position of the substituent, the quinone methide formation can result in the elimination of HX from the naphthazarin. The alkylating agent quinone methide is believed to act as Michael acceptor of a biologically important nucleophile (Nu: DNA, protein, carbohydrate, etc.,) which may eventually lead to cell death. Comparing C1 and C2, it was found that C2 exhibited higher % cell inhibition on SiHa, KB, and HCT 116 cancer cell lines. These results indicate that the presence of an acetyl group may be critical for the cytotoxicity. It is believed that the acetyl group in C2 contributes significantly to its cytotoxicity as it showed 100.0 ± 0.2% cell inhibition at a concentration of 0.30 μM on HCT116 cell line [Table S1]. Toxic and electrophilic C1 and C4 carbonyl carbons of quinones react preferentially with the genetic material of macromolecule due to their high susceptibility toward a nucleophilic attack clarify the underlying chemistry involved in this critical event.
| Conclusion|| |
The use of traditional medicinal plants has become essential as most of the anticancer compounds are identified and isolated from plants. Various genus of medicinal plants have been explored for isolation of chemotherapy drugs. In this study, two compounds hydroxyisovalerylshikonin and acetylshikonin isolated from A. euchroma, an important traditional Chinese medicinal herb were tested for inhibitory effect against SiHa, KB, HCT116 human cancer cell lines. Both the compounds showed promising % cell inhibition against HCT116 cell lines.
We thank Director IHBT Dr. Sanjay Kumar for all the necessary facilities provided by the institute. We thank the National Centre for Cell Science, Pune, and Indian Institute of Integrative Medicine, Jammu. This is IHBT communication number - 4137.
Financial support and sponsorship
NS gratefully thanks and acknowledges the Department of Science and Technology New Delhi for financial support (SR/WOS-A/CS-08/2014).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Chukavina AP. A new genus of Boraginaceae
. Dokl Akad Nauk Tadzh SSR 1974;17:63-6.
Singh H, Chauhan R, Raina R. Population structure, ecological features and associated species of Arnebia euchroma
. J Pharmacogn Phytochem 2017;6:2005-7.
The Committee of Pharmacopeia of PLA. Beijing: Press of Chemistry and Industry; 2005. p. 238.
Fu SL, Xiao PG. A study on the naphthoquinone pigments from Arnebia euchroma
. Zhong CaoYao 1986;17:2.
Yao XS, Ebizuka Y, Noguchi H, Kiuchi F, Shibuya M, Iitaka Y, et al.
Biologically active constituents of Arnebia euchroma
: Structures of new monoterpenylbenzoquinones: Arnebinone and arnebifuranone. Chem Pharm Bull (Tokyo) 1991;39:2962-4.
Yao XS, Ebizuka Y, Noguchi H, Kiuchi F, Shibuya M, Iitaka Y, et al.
Biologically active constituents of Arnebia euchroma
: Structure of arnebinol, an ansa-type monoterpenylbenzenoid with inhibitory activity on prostaglandin biosynthesis. Chem Pharm Bull (Tokyo) 1991;39:2956-61.
Zhang HJ, Liao MC, Xuan LJ, Guo JX. A contraceptive constituent from Arnebia euchroma
(Royle) Johnst. Nat Prod Res Dev 2002;14:1-4.
Yang MH, Blunden G, O'Neill MJ, Lewis JA. Tormentic acid and 2α-hydroxyursolic acid from Arnebia euchroma
. Planta Med 1992;58:227.
Röder E, Rengel-Mayer B. Pyrrolizidine alkaloids from Arnebia euchroma
*. Planta Med 1993;59:192.
Assimopoulou AN, Karapanagiotis I, Vasiliou A, Kokkini S, Papageorgiou VP. Analysis of alkannin derivatives from Alkanna species by high-performance liquid chromatography/photodiode array/mass spectrometry. Biomed Chromatogr 2006;20:1359-74.
Singh B, Sharma MK, Meghwal PR, Sahu PM, Singh S. Anti-inflammatory activity of shikonin derivatives from Arnebia hispidissima
. Phytomedicine 2003;10:375-80.
Sasaki K, Abe H, Yoshizaki F. In vitro
antifungal activity of naphthoquinone derivatives. Biol Pharm Bull 2002;25:669-70.
Assimopoulou AN, Boskou D, Papageorgiou VP. Antioxidant activities of alkannin, shikonin and Alkanna tinctoria
root extracts in oil substrates. Food Chem 2004;87:433-8.
Kourounakis AP, Assimopoulou AN, Papageorgiou VP, Gavalas A, Kourounakis PN. Alkannin and shikonin: Effect on free radical processes and on inflammation – A preliminary pharmacochemical investigation. Arch Pharm (Weinheim) 2002;335:262-6.
Davis PH. Flora of Turkey and the East Aegean Islands. Edinburgh: Edinburg University; 1978. p. 414.
An S, Park YD, Paik YK, Jeong TS, Lee WS. Human ACAT inhibitory effects of shikonin derivatives from Lithospermum erythrorhizon
. Bioorg Med Chem Lett 2007;17:1112-6.
Kajimoto S, Hori M, Manabe H, Masuda Y, Shibayama-Imazu T, Nakajo S, et al
. A tyrosine kinase inhibitor β-hydroxyisovalerylshikonin, induced apoptosis in human lung cancer DMS114 cells through reduction of dUTP nucleotidohydrolase activity. Biochim Biophys Acta Mol Basis Dis 2008;1782:41-50.
Ahn BZ, Baik KU, Kweon GR, Lim K, Hwang BD. Acylshikonin analogues: Synthesis and inhibition of DNA topoisomerase-I. J Med Chem 1995;38:1044-7.
Plyta ZF, Li T, Papageorgiou VP, Mellidis AS, Assimopoulou AN, Pitsinos EN, et al.
Inhibition of topoisomerase I by naphthoquinone derivatives. Bioorg Med Chem Lett 1998;8:3385-90.
Lu Q, Liu W, Ding J, Cai J, Duan W. Shikonin derivatives: Synthesis and inhibition of human telomerase. Bioorg Med Chem Lett 2002;12:1375-8.
Yang F, Yi F, Chen Y, Duan W, Zhang C, Hong Z, et al
. SH-7, a new synthesized shikonin derivative, exerting its potent antitumor activities as a topoisomerase inhibitor. J Int J Cancer 2006;119:1184-93.
Driscoll JS, Hazard GF, Wood HB Jr., Goldin A Jr. Structure-antitumor activity relationships among quinone derivatives. Cancer Chemother Rep Part 2 1974;4:1-362.
Kumar R, Sharma S, Sharma S, Kumari A, Kumar D, Nadda G, et al
. Chemical composition, cytotoxicity and insecticidal activities of Acorus calamus
accessions from the Western Himalayas. Ind Crops Prod 2016;94:520-7.
Shin SY, Yong Y, Hong DS, Lee DH, Lee DY, Lee YH. Identification of flavonoids from Eriodictyon californicum
and their cytotoxicity against HCT116 colon cancer cells. J Korean Soc Appl Biol Chem 2015;1:77-81.
Yadav R, Kumar D, Kumari A, Yadav SK. Encapsulation of podophyllotoxin and etoposide in biodegradable poly-D, L-lactide nanoparticles improved their anticancer activity. J Microencapsul 2014;31:211-9.
Singla P, Dalal P, Kaur M, Arya G, Nimesh S, Singh R, et al.
Bile acid oligomers and their combination with antibiotics to combat bacterial infections. J Med Chem 2018;61:10265-75.
van Asten SD, Raaben M, Nota B, Spaapen RM. Secretome screening reveals fibroblast growth factors as novel inhibitors of viral replication. J Virol 2018;92. pii: e00260-18.
Damianakos H, Kretschmer N, Sykłowska-Baranek K, Pietrosiuk A, Bauer R, Chinou I. Antimicrobial and cytotoxic isohexenylnaphthazarins from Arnebia euchroma
(Royle) Jonst. (Boraginaceae
) callus and cell suspension culture. Molecules 2012;17:14310-22.
Kim DJ, Lee JH, Park HR, Choi YW. Acetylshikonin inhibits growth of oral squamous cell carcinoma by inducing apoptosis. Arch Oral Biol 2016;70:149-57.
Figat R, Zgadzaj A, Geschke S, Sieczka P, Pietrosiuk A, Sommer S, et al.
Cytotoxicity and antigenotoxicity evaluation of acetylshikonin and shikonin. Drug Chem Toxicol 2018;21:1-8.
Jing Y, Shaoshun L, Niu W, Wei W, Wen Z, Xinzhi H. Application of Beta Hydroxyisovaleryl Shikonin in Medicament Production. Google Patents; 2013.
Ahn BZ, Song GY, Baik KU, Sok DE. Cytotoxicity of acylshikonin analogues against L1210 cells and their antitumor activity against sarcoma tumors. Korean J Med Chem 1996;6:98-109.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]